X-ray crystallographic analyses of the structures of two heme proteins : a thesis submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in the Institute of Molecular BioSciences at Massey University, New Zealand

Abstract

During human development three embryonic hemoglobins are synthesised prior to formation of the placenta. These hemoglobins function to scavenge oxygen from the mother's interstitial fluid enabling embryonic respiration. The human Gower II embryonic haemoglobin (α2ε2) has been crystallized in its carbonmonoxy form, and its structure determined by X-ray crystallography. The structure was solved by molecular replacement and refined at 2.9 Å. The Gower II hemoglobin tetramer is intermediate between the adult hemoglobin R and R2 states, though closer to R2. The tertiary structure of the α subunit is essentially identical when compared to that found in the adult (α2β2) and fetal (α2γ2) hemoglobins. The embryonic ε subunit has a very similar structure to the homologous adult β and fetal γ subunits, although with small differences at the N-terminus and in the A helix. Amino acid substitutions can be identified that may play a role in the altered response of the Gower II haemoglobin to allosteric effectors, in particular chloride ions. Nitrite reductase from Pseudomonas stutzeri is a periplasmic heme enzyme responsible for the reduction of nitrite to nitric oxide. This reaction is the second step in the bacterial denitrification pathway, during which nitrate acts as the terminal electron acceptor for anaerobic respiration and is consequently reduced to nitrogen gas. Nitrite reductase from Pseudomonas stutzeri JM300 has been crystallized in the oxidised state and X-ray diffraction data collected to a resolution of 2.8 Å. The structure has been solved by the method of molecular replacement. The structure of the enzyme is dimeric, with each monomer comprised of two domains. The smaller N-terminal domain covalently binds a c heme group within an all α-helical fold similar to that of the class l c-type cytochromes. The larger C-terminal domain consists of an eight-bladed β-propeller structure that coordinates a d1 heme, a cofactor unique to this class of enzyme. The relative positions of the two domains, and hence the orientations of the bound heme groups are markedly different compared to homologous enzymes from other species.